1. Field of the Invention
The present invention relates to a method of quantitatively analyzing the concentration or activity of an analyte contained in a whole blood sample by using a dry analysis element.
2. Prior Art
It is important to determine the content or activity of an analyte in a living body fluid, particularly in blood, for example, for the diagnosis of diseases or for monitoring the course of remedy. For such purpose, dry chemical analysis elements (or strips) have been increasingly used in recent years, since they are economical and operable with ease to give test results rapidly. The dry chemical analysis is a method wherein a test strip or multi-layered analysis element is used in the dry state different from the conventional chemical analysis conducted in a wet system. Dry analysis elements are disclosed, for example, in Japanese Patent Publication No. 21677/1978 (corresponding to U.S. Pat. No. 3,992,158), Unexamined Japanese Patent Publication No. 164356/1980 (corresponding to U.S. Pat. No. 4,292,272) and Unexamined Japanese Patent Publication No. 222769/1985 (corresponding to EP 0 162 302A). The dry multi-layered analysis element comprises, for example, a transparent support, a reagent layer and a spreading layer. The transparent support is made of, for example, a thin light-permeable and water-impermeable organic polymer sheet. The reagent layer is coated over the transparent support and contains a reagent composition which reacts with the analyte in a liquid sample taken from a living body to give some change in signal, for example, change in color density due to coloring by a formed dye, the color change being in proportion to the quantity of analyte contained in the sample. The spreading layer spreads the liquid sample spotted thereon uniformly so that a spread area is in proportion to the volume of the spotted liquid sample.
When a dry multi-layered analysis element is used for the quantitative analysis, a proper volume of a liquid sample is spotted on the surface of the spreading layer by dropping or otherwise applying the liquid sample. The liquid sample is uniformly spread within the spreading layer to be passed to the reagent composition layer where the analyte contained in the sample reacts with the reagent composition to exhibit a change in some signal. The analysis element is held at a constant temperature for a sufficient time period for completing the reaction (incubation), and then the change in some signal is detected. When the signal is a color change due to formation of some dye, the reagent composition layer is irradiated with a light from the transparent support side and the optical density of the reflected light is measured at a certain wavelength. The content (concentration or activity) of the analyte may be colorimetrically determined by comparing the thus detected signal change with a calibration curve (or table) which is preliminarily drawn by plotting the interrelation between the signal change and the content of analyte in the liquid sample. The signal change may be the absolute value of a parameter after the lapse of a predetermined time from the time at which the liquid sample is spotted, or may be the changing rate within a unit time.
However, when a whole blood sample is to be analyzed by the use of most of the conventionally proposed or commonly used dry analysis elements, it becomes necessary to remove blood cells from the whole blood sample to separate blood serum or plasma, generally by means of centrifugal separation, since whole blood cannot be used directly as a liquid sample in some dry analysis elements. For this reason, irrespective of the merits of the analysis in which dry analysis elements are used, i.e. easy handling, rapidity and economical saving attainable by the use of the dry analysis element, practical utility thereof is reduced seriously since it requires considerable labours and time with the need of using an expensive equipment to remove blood cells from the whole blood sample.
In quantitative analysis of an analyte contained in a whole blood sample by using a dry analysis element, it is required (1) to remove blood cell components, particularly red blood cells, from the sample by any means or reaction, and in addition (2) to prevent the red metters (hemoglobin) of the separated blood cells from hindering the operation of measuring the optical density. Japanese Patent Publication No. 21677/1978 (corresponding to U.S. Pat. No. 3,992,158) discloses that a filtering layer is disposed within an analysis element for separating and removing blood cells contained in whole blood. However, it requires longer time to remove blood cells from whole blood to separate blood serum or plasma by the filtering layer. Furthermore, there is a fear that a portion of the analyte to be analyzed might be lost during the filtering operation or hemolysis of red blood cells might occur to result in inaccurate analysis.
On the other hand, Unexamined Japanese Patent Publication No. 138756/1987 (correponding to EP 0 226 465A) proposes a dry analysis element conveniently used for the quantitative analysis of an analyte in a whole blood sample, in which removal of blood cells in the whole blood sample is accelerated so that the analyte in the separated plasma is rapidly spread into the reagent composition layer. However, the analysis element of this prior proposal has a disadvantage that the results of analysis are affected by the hematocrit values of individual blood samples to give different test results even when the contents of analyte in respective samples are same. Accordingly, it is difficult to perform accurate analysis when such an analysis element is used. Unexamined Japanese Patent publication No. 54354/1989 (corresponding to EP 0 302 287A) proposes the provision of a light-shielding layer containing a white pigment to shield the light irradiated for the purpose of determination so as to eliminate the influence of red matters contained in red blood cells. It is estimated that the provision of such a light-shielding layer is effective for excluding the influence by the red matters contained in red blood cells to improve the accuracy of the determination operation. However, the particle size and dispersion of the pigment particles must be precisely controlled in order to adjust the light-shielding rate, leading to complication of the production steps and increase in production cost. In addition, there is found a case where separation of blood cell components cannot be performed sufficiently or sufficient amount of separated plasma is not fed into the reagent composition layer.